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Purification and characterization of a protease from Thermophilic bacillus strain HS08
H Guangrong, Y Tiejing, H Po, J Jiaxing
African Journal of Biotechnology , 2006,
Abstract: The purification and characterization of a thermophilic neutral protease from Thermophilic bacillus strain HS08, originally isolated from a soil sample collected from the Tulufan Crater of China, is presented in this paper. The purification steps included ammonium sulfate precipitation, with columns of DEAE-Sepharose anion exchange chromatography and Sephacryl S-100HR on AKTA purifier 100 protein liquid chromatography. The method gave a 4.25 fold increase of the specific activity and had a yield of 5.1%. The molecular weight of the protease was found to be around 30.9 kDa by SDS-PAGE technique. The optimal pH and optimal temperature of the protease were at pH 7.5 and 65oC, respectively. The protease was found stable during the 1 h incubation at 50°C. The protease activity showed wide range of variation in the presence of different reagents: it was inhibited remarkably by EDTA or PMSF and was almost activated by 2 mM Zn2+, even though it was only marginally inhibited by other inhibitors. We concluded that the protease was a Zn2+-acitived serine protease. Substrates specificity tests indicated that azocasein was the best substrate among the three substrates tested (azocasein, casein, and BSA).
Serine protease Jurassic Park
Cathy Holding
Genome Biology , 2003, DOI: 10.1186/gb-spotlight-20030903-01
Abstract: Wouters et al. inferred ancestral sequences from parsimony analysis of a multiple alignment of 56 immune defense protease (IDP) sequences and constructed a synthetic gene to express the recombinant protein that they called Stemzyme-IDP-β. Using angiotensin II as substrate for kinetic and binding studies, the authors observed high catalytic efficiency and broad substrate specificity and a tolerance to mutation at the binding site, with different mutations resulting in activities similar to some of the synthetic enzyme's descendents. They suggest that a form of reverse evolution must have occurred that provided a mechanism for enzymes with narrow specificity to evolve from enzymes with already narrow specificity."Our findings... suggest that once a narrow primary specificity had become established, the further generation of diversity required a reversion to the presumed original state. That is, despecialization or evolution-in-reverse was required for further diversification," the authors conclude.
Serine Protease Autotransporters of Enterobacteriaceae (SPATEs): Biogenesis and Function  [PDF]
Nathalie Dautin
Toxins , 2010, DOI: 10.3390/toxins2061179
Abstract: Serine Protease Autotransporters of Enterobacteriaceae (SPATEs) constitute a large family of proteases secreted by Escherichia coli and Shigella. SPATEs exhibit two distinct proteolytic activities. First, a C-terminal catalytic site triggers an intra-molecular cleavage that releases the N-terminal portion of these proteins in the extracellular medium. Second, the secreted N-terminal domains of SPATEs are themselves proteases; each contains a canonical serine-protease catalytic site. Some of these secreted proteases are toxins, eliciting various effects on mammalian cells. Here, we discuss the biogenesis of SPATEs and their function as toxins.
The serine protease inhibitors and plant-insect interaction
Mendoza-Blanco,Werner; Casaretto,José A;
Idesia (Arica) , 2012, DOI: 10.4067/S0718-34292012000100015
Abstract: plants respond to a physical injury or biological attack by producing, among other compounds, an arsenal of defense proteins, secondary metabolites and phytohormones, all necessary for plant survival. defense proteins include the group of serine protease inhibitors (spi), proteins that interact with the active site of their target enzymes. the activity of these protease inhibitors has been exploited to combat insect pests. spi is an interesting alternative to produce plants with improved resistance characters through selection in the field or expressing their genes in sensitive plants by genetic engineering. the alternative that these natural products offer makes them valuable for the control of several crop pests.
New insights into the evolution of subtilisin-like serine protease genes in Pezizomycotina
Juan Li, Li Yu, Jinkui Yang, Linqian Dong, Baoyu Tian, Zefen Yu, Lianming Liang, Ying Zhang, Xu Wang, Keqin Zhang
BMC Evolutionary Biology , 2010, DOI: 10.1186/1471-2148-10-68
Abstract: Phylogenetic analysis of 189 subtilisin-like serine protease genes from Pezizomycotina suggests five strongly-supported monophyletic clades. The subtilisin-like serine protease genes previously identified or presumed as endocellular proteases were clustered into one clade and diverged the earliest in the phylogeny. In addition, the cuticle-degrading protease genes from entomopathogenic and nematode-parasitic fungi were clustered together, indicating that they might have overlapping pathogenic mechanisms against insects and nematodes. Our experimental bioassays supported this conclusion. Interestingly, although they both function as cuticle-degrading proteases, the subtilisin-like serine protease genes from nematode-trapping fungi and nematode-parasitic fungi were not grouped together in the phylogenetic tree. Our evolutionary analysis revealed evidence for positive selection on the subtilisin-like serine protease genes of the nematode-trapping fungi.Our study provides new insights into the evolution of subtilisin-like serine protease genes in Pezizomycotina. Pezizomycotina subtilisins most likely evolved from endocellular to extracellular proteases. The entomopathogenic and nematode-parasitic fungi likely share similar properties in parasitism. In addition, our data provided better understanding about the duplications and subsequent functional divergence of subtilisin-like serine protease genes in Pezizomycotina. The evidence of positive selection detected in the subtilisin-like serine protease genes of nematode-trapping fungi in the present study suggests that the subtilisin-like serine proteases may have played important roles during the evolution of pathogenicity of nematode-trapping fungi against nematodes.Subtilisin-like serine proteases play an important role in the pathogenicity of pathogenic fungi. By using subtilisin-like serine proteases, pathogenic fungi disrupt the physiological integrity of the hosts during penetration and colonization [1,2]. Previous s
Prevalence, Biogenesis, and Functionality of the Serine Protease Autotransporter EspP  [PDF]
André Weiss,Jens Brockmeyer
Toxins , 2013, DOI: 10.3390/toxins5010025
Abstract: Enterohemorrhagic E. coli (EHEC) causes severe diseases in humans worldwide. One of its virulence factors is EspP, which belongs to the serine protease autotransporters of Enterobacteriaceae (SPATE) family. In this review we recapitulate the current data on prevalence, biogenesis, structural properties and functionality. EspP has been used to investigate mechanistic details of autotransport, and recent studies indicate that this transport mechanism is not autonomous but rather dependent on additional factors. Currently, five subtypes have been identified (EspPα-EspPε), with EspPα being associated with highly virulent EHEC serotypes and isolates from patients with severe disease. EspPα has been shown to degrade major proteins of the complement cascade, namely C3 and C5 and probably interferes with hemostasis by cleavage of coagulation factor V. Furthermore, EspPα is believed to contribute to biofilm formation perhaps by polymerization to rope-like structures. Together with the proteolytic activity, EspPα might ameliorate host colonization and interfere with host response.
Regulation of Adrenal Aldosterone Production by Serine Protease Prostasin
Takehiro Ko,Yutaka Kakizoe,Naoki Wakida,Manabu Hayata,Kohei Uchimura,Naoki Shiraishi,Taku Miyoshi,Masataka Adachi,Shizuka Aritomi,Tomoyuki Konda,Kimio Tomita,Kenichiro Kitamura
Journal of Biomedicine and Biotechnology , 2010, DOI: 10.1155/2010/793843
Abstract: A serine protease prostasin has been demonstrated to have a pivotal role in the activation of the epithelial sodium channel. Systemic administration of adenovirus carrying human prostasin gene in rats resulted in an increase in plasma prostasin and aldosterone levels. However, the mechanism by which the elevation of prostasin levels in the systemic circulation stimulated the plasma aldosterone levels remains unknown. Therefore, we examined if prostasin increases the aldosterone synthesis in a human adrenocortical cell line (H295R cells). Luciferase assay using CYP11B2 promoter revealed that prostasin significantly increased the transcriptional activity of CYP11B2. Prostasin significantly increased both CYP11B2 mRNA expression and aldosterone production in a dose-dependent manner. Surprisingly, treatment with camostat mesilate, a potent prostasin inhibitor, had no effect on the aldosterone synthesis by prostasin and also a protease-dead mutant of prostasin significantly stimulated the aldosterone production. A T-type/L-type calcium channel blocker and a protein kinase C (PKC) inhibitor significantly reduced the aldosterone synthesis by prostasin. Our findings suggest a stimulatory effect of prostasin on the aldosterone synthesis by adrenal gland through the nonproteolytic action and indicate a new role of prostasin in the systemic circulation.
A secreted serine protease of Paracoccidioides brasiliensis and its interactions with fungal proteins
Juliana A Parente, Sílvia M Salem-Izacc, Jaime M Santana, Maristela Pereira, Clayton L Borges, Alexandre M Bail?o, Célia MA Soares
BMC Microbiology , 2010, DOI: 10.1186/1471-2180-10-292
Abstract: A cDNA (Pbsp) encoding a secreted serine protease (PbSP), was isolated from a cDNA library constructed with RNAs of fungal yeast cells recovered from liver of infected mice. Recombinant PbSP was produced in Escherichia coli, and used to develop polyclonal antibodies that were able to detect a 66 kDa protein in the P. brasiliensis proteome. In vitro deglycosylation assays with endoglycosidase H demonstrated that PbSP is a N-glycosylated molecule. The Pbsp transcript and the protein were induced during nitrogen starvation. The Pbsp transcript was also induced in yeast cells infecting murine macrophages. Interactions of PbSP with P. brasiliensis proteins were evaluated by two-hybrid assay in the yeast Saccharomyces cerevisiae. PbSP interacts with a peptidyl prolyl cis-trans isomerase, calnexin, HSP70 and a cell wall protein PWP2.A secreted subtilisin induced during nitrogen starvation was characterized indicating the possible role of this protein in the nitrogen acquisition. PbSP interactions with other P. brasiliensis proteins were reported. Proteins interacting with PbSP are related to folding process, protein trafficking and cytoskeleton reorganization.Serine protease is a class of peptidases widely distributed in all domains of life that use a serine residue at the active site to cleave peptides [1]. Serine proteases are associated with virulence and nutrient cycling in many pathogens. In the human pathogen Trichophyton rubrum seven serine proteases genes were detected, two of them encoding products able to cleave keratin, suggesting the importance of these proteases in the invasion process in the human host [2]. Also, a secreted serine protease from Microsporum canis was described. A serine protease inhibitor, as well as a monoclonal antibody directed to the protein inhibited fungal adherence to reconstructed interfollicular feline epidermis [3]. In the entomophatogenic fungus Magnaporthe grisea, the SPM1 serine protease is positively regulated during nitrogen sta
The role of Serine Proteases and Serine Protease Inhibitors in the migration of Gonadotropin-Releasing Hormone neurons
Paola T Drapkin, Denis Monard, Ann-Judith Silverman
BMC Developmental Biology , 2002, DOI: 10.1186/1471-213x-2-1
Abstract: Affigel blue beads were used to deliver a serine protease inhibitor, protease nexin-1 (PN-1), and a target protease, trypsin, to the olfactory epithelium coincident with initiation of GnRH neuronal migration. PN-1 inhibited neuronal migration while trypsin accelerated their transit into the CNS. Prior to initiation of migration, neither PN-1 nor trypsin altered the timing of neuronal exit. Trypsin did, however, accelerate the timing of neuronal crossing into the nerve-forebrain junction.These data support the hypothesis that protease activity modulates neuronal movements across barriers. Moreover, the data suggest, for the first time, that aspects of GnRH neuronal migration may be cell autonomous but modulated by ECM alterations.A key component regulating neuronal migration is the appropriate spatio-temporal expression of extracellular matrix (ECM) molecules which contribute to the highway along which neurons travel. Proteins, such as serine proteases and their inhibitors, could alter the quality of this highway and thus play critical roles in migratory processes [1]. Members of the serine protease inhibitor superfamily, or serpins, act by binding to and permanently inactivating their target protease(s). One member of this family, protease nexin-1 (PN-1), was first described by Monard et al.[2]. Though initially characterized for its ability to stimulate neurite outgrowth [2-5], PN-1 also modulates neuronal migration as exemplified by Lindner et al.[6]. Their studies demonstrate that PN-1 slows granule cell movement from the external to internal layers in postnatal mouse cerebellar slice cultures.Interestingly, this inhibition of migration by protease inhibitors may be balanced by the stimulation of migration via proteases. For example in vitro addition of plasmin, a serine protease, accelerates the migration of neuroblastoma cells through a matrigel base by a factor of five [7]. The subsequent addition of aprotinin, a plasmin inhibitor, decreases the migratory popu
Production and properties of an extracellular protease from thermophilic Bacillus sp
Nascimento, Wellingta Cristina Almeida do;Martins, Meire Lelis Leal;
Brazilian Journal of Microbiology , 2004, DOI: 10.1590/S1517-83822004000100015
Abstract: protease production by thermophilic bacillus sp strain smia-2 cultivated in liquid cultures containing trisodium citrate reached a maximum in 9h, with levels of 1.93u/mg protein. the microorganism utilized several carbon sources for the production of protease. starch was the best substrate, followed by trisodium citrate, citric acid and sucrose. among the various organic and inorganic nitrogen sources, ammonium nitrate was found to be the best. studies on the protease characterization revealed that the optimum temperature of this enzyme was 60oc. the enzyme was stable for 2h at 30oc, while at 40oc and 80oc, 14% and 84% of the original activities were lost, respectively. the optimum ph of the enzyme was found to be 8.0. after incubation of crude enzyme solution for 24h at ph 5.5, 8.0 and 9.0, a decrease of about 51%, 18% and 66% of its original activity was observed respectively. a stronger inhibitory effect was observed in the presence of k+, hg2+and cu2+. hg+ resulted in the complete loss of activity at 1mm concentrations. activity was stimulated by mn2+ and ca+2, indicating that these ions had a functional role in the molecular structure of the enzyme.
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